Methods for determining antiphospholipid antibodies

ABSTRACT

The invention provides phospholipid coated particles capable of specifically binding antiphospholipid antibodies and a method for preparing such particles. Methods are also provided for determining antiphospholipid antibodies in a serum or plasma. Also provided are methods for isolating antiphospholipid antibodies from a fluid and for raising specific antiphospholipid antibodies.

This application is a divisional of U.S. Ser. No. 08/129,593, filed Sep.30, 1993, now U.S. Pat. No. 5,361,070, which is a divisional of U.S.Ser. No. 07/905,562, filed Jun. 29, 1992, which is now abandoned.

BACKGROUND

This invention relates to methods for detecting and measuring antibodiesto phospholipids.

Antibodies to phospholipids have been implicated in a variety ofdiseases or clinical conditions including arterial and venousthrombosis, recurrent fetal loss, thrombocytopenia, pulmonary embolism,coronary thrombosis, cerebral thrombosis, livedo reticularis and HIVinfection.

It is therefore important to have a convenient and reproducible clinicalmethod for detecting and measuring antiphospholipid antibodies insamples from patients.

A variety of phospholipids can give rise to antibodies in humans andantibodies specific to cardiolipin, phosphatidylinositol,phosphatidylserine and phosphatidylethanolamine, for example, have beenfound.

Not all of these antibodies are necessarily present in every diseaseassociated with anti-phospholipid antibodies. It is therefore desirableto be able to determine easily antibodies to various specificphospholipids.

Furthermore, antibodies to a particular phospholipid may be IgG, IgM orIgA type immunoglobulins and not all types may be present in allconditions; for example, IgA antibodies have been linked to adrenalinsufficiency (Al-Momen et al., (1991), Thromb. Res., Vol 64, p. 571)and high levels of IgG antiphospholipid antibodies have been noted insystemic lupus erythematosus (Quamar, T. et al., (1990), ArthritisRheum, Vol 33, p. 501).

It is therefore desirable to be able to distinguish antibodies ofseveral immunoglobulin classes.

Presently available commercial methods for determining antiphospholipidantibodies are based on enzyme-linked immunosorbent assays (ELISA).Commercially available kits use cardiolipin as substrate and thereforedetect only anti-cardiolipin antibodies.

Radioimmunoassays have also been used to measure anti-phospholipidantibodies but these have all the drawbacks associated with the use ofradioactive materials.

A flow cytometric assay has also been reported for determininganti-cardiolipin antibodies (Pirruccello et al; (1990), J. Clin. Lab.Anal., vol. 4, p. 236). This assay utilises liposomes as carriers of thephospholipids. The difficulty of controlling liposome size makesstandardization of the liposomes a problem. In addition, liposomes aregenerally composed of more than one phospholipid to lend stability tothe liposome.

The methods presently available for determination of antiphospholipidantibodies are limited in their flexibility and convenience,particularly when one considers the increasing clinical need fordetermination of antibodies of different immunoglobulin classes andspecific to particular phospholipids.

SUMMARY OF THE INVENTION

In accordance with one aspect of the invention, a method is provided fordetermining antiphospholipid antibodies in a fluid comprising the stepsof:

(a) providing phospholipid coated particles coated with at least onephospholipid for which antibodies are to be determined;

(b) contacting the fluid with the coated particles to permit binding ofthe antibodies to the particles; and

(c) determining the antiphospholipid antibodies bound to the particles.

In accordance with a further aspect of the invention, a method isprovided for isolating from a fluid antibodies to a phospholipid themethod comprising:

(a) contacting the fluid with phospholipid coated particles wherein thephospholipid is that for which antibodies are to be isolated, to permitbinding of the antibodies to the particles;

(b) separating the particles from the residual solution;

(c) recovering the anti-phospholipid antibodies from the particles.

In accordance with a further aspect of the invention, a method isprovided for producing antiphospholipid antibodies specific to aphospholipid comprising providing particles treated with thephospholipid to permit binding of the phospholipid to the particles andinjecting the treated particles subcutaneously into a suitable animal.

In accordance with a further aspect of the invention, a method isprovided for preparing phospholipid coated particles comprising:

(a) contacting particles with a solution of a phospholipid at aneffective temperature to permit binding of the phospholipid to theparticles; and

(b) contacting the particles with a blocking agent at an effectivetemperature to permit blocking of non-specific binding sites on theparticles.

In accordance with a further aspect of the invention, particles areprovided which are coated with at least one phospholipid, the coatedparticles being able to bind specifically antibodies to the at least onephospholipid.

SUMMARY OF DRAWINGS

The invention, as exemplified by preferred embodiments, is describedwith reference to the drawings in which:

FIG. 1 shows: (a) the time course of antibody binding and (b) the effectof temperature on non-specific binding.

FIG. 2 is a scatter histogram showing bead size discrimination by flowcytometry.

FIG. 3 is a standard curve in APL units.

FIG. 4 is a standard curve in GPL units.

FIG. 5 shows normal ranges of antiphospholipid antibodies in humanserum, determined by the method of the invention.

FIG. 6 shows antiphospholipid antibody levels in seven patients.

FIG. 7 shows a comparison of serum anticardiolipin antibody levelsdetermined by ELISA and by the method of the invention.

DETAILED DESCRIPTION OF THE INVENTION

In accordance with a first embodiment, the subject invention providesnovel phospholipid coated particles which show specific binding ofantiphospholipid antibodies, are easily prepared, stable and can beconveniently manipulated and employed in a variety of methods fordetermining antiphospholipid antibodies. The versatility of thephospholipid coated particles of the invention provides assays which canconveniently identify antiphospholipid antibodies of different classesand different phospholipid specifities, and provides for quantitation ofthese antibodies, by methods which are reproducible and sensitive andsuitable for clinical diagnostic purposes.

In accordance with a further embodiment of the invention, a method isprovided for preparing such phospholipid coated particles, for examplemicrospheres or beads.

The inventors have found that polystyrene microspheres or beads canunexpectedly be coated with a phospholipid by relatively gentletechniques. Although microspheres coated with proteins have previouslybeen described, the hydrophobic nature of phospholipids makes itdifficult for these to stick to the charged microsphere surface.

In accordance with a preferred embodiment, polystyrene microspheres aretreated with a solution of the desired phospholipid in ethanol in thedark for at least about twelve hours at an effective temperature, topermit binding of the phospholipid. In an especially preferredembodiment, phospholipid treatment is carried out at a temperature inthe range of about 0° to about 4°. As will be appreciated by thoseskilled in the art, the microspheres can be coated with phospholipid athigher temperatures but as the treatment temperature is raised aboveabout 4° C., there is increasing damage to phospholipid molecules sothat when the coated microspheres are used for assay of antiphospholipidantibodies, as will be described, less than optimal results areobtained; for example, coating of the microspheres with phospholipid at22° C. produces broad peaks on flow cytometry and reduces specificityand sensitivity of the assay.

After treatment with phospholipid, the microspheres can be employed toraise specific antiphospholipid antibodies as will be described. Themicrospheres can also be stored at this stage, as described in Example1.

When the microspheres are to be employed for determination ofantiphospholipid antibodies or for isolation of antibodies, they aretreated with a blocking agent to reduce non-specific binding ofimmunoglobulins and other potentially interfering proteins. Inaccordance with a preferred embodiment, the microspheres are treatedwith 10% fetal calf serum as blocking agent for about 15 to about 60minutes at a temperature in the range of about 4° C. to about 37° C. Itis especially preferred to carry out blocking at about 37° C. as thisgives the lowest levels of non-specific binding when the microspheresare used to determine antiphospholipid antibodies. Blocking at lowertemperatures tends to increase the level of non-specific binding to themicrospheres in the assay.

The phospholipid coated microspheres are generally cooled after blockingtreatment to a temperature of about 0° C. to about 4° C., since this isthe temperature range at which they are optimally stored or employed inthe antiphospholipid antibody assay.

It is believed that the incubation of the phospholipid treated beadswith the blocking agent at an elevated temperature renders thephospholipid more mobile about the surface of the bead, resulting inefficient exposure of the bead surface sites responsible fornon-specific binding of proteins. The rapid cooling may render thephospholipid less mobile about the bead, effectively locking thephospholipid molecules in place, with the majority of the nonspecificbinding sites blocked.

In this specification, the terms `coated particles`, `phospholipidcoated particles`, `coated microspheres or beads` and the like are usedto mean particles, microspheres or beads coated with phospholipid andtreated with blocking agent. Where particles, microspheres or beads withbound phospholipid but without blocking treatment are referred to, thisis specifically indicated.

Polystyrene microspheres are obtainable in a variety of sizes and arepreferred for preparation of the phospholipid coated particles of theinvention. Other suitable solid particulate supports will be known tothose skilled in the art and include particles of suitable shapecomposed of a material capable of holding a charge such aspolypropylene, polyethylene, acrylonitrile, polycarbonate ornitrocellulose or magnetic beads. The shape of the particle should becompatible with the needs of the method ultimately employed to determineantiphospholipid antibodies once these are bound to the phospholipidcoated particle. For example, if flow cytometry is to be employed, theparticles should be generally spherical. The size of particle employedshould also be suitable for the detection method to be employed. Forexample, microspheres of diameter from about 0.8 μm to about 10 μm aresuitable for flow cytometry whereas larger or smaller particles may berequired for other assay techniques e.g., around 0.06 μm diameter forlatex enhanced laser nephelometry or around 15 μm for latex beadagglutination.

Adult bovine serum has also been found to be suitable as blocking agentfor work with antiphospholipid antibodies, as described by Harris et al.(Clin, Exp. Immunol. (1987), vol. 68, p. 215) whereas certain otherproteins are unsuitable. A suitable blocking agent should provide lowlevels of non-specific binding when the microspheres are used forantiphospholipid antibody assay.

One of the advantages of using microspheres such as polystyrenemicrospheres as particulate supports to prepare phospholipid coatedparticles is their availability in a variety of sizes.

In accordance with a further embodiment of the invention, microspheresof different sizes are selected and each size of microspheres is coatedwith a different phospholipid, followed by treatment with blockingprotein, as described in Example 1.

The coated microspheres may be easily distinguished by virtue of theirsize differences, allowing for rapid differentiation of antibodies todifferent phospholipids, as will be described. As will be understood bythose skilled in the art, microspheres of a single size may be coatedwith a mixture of phospholipids but this will provide for assay of totalantiphospholipid antibodies only and not for any differentiation.

The phospholipid coated microspheres of the invention provide aconvenient antigen-presenting device which may be employed along with avariety of detection procedures to detect and to determineantiphospholipid antibodies.

In accordance with a further preferred embodiment of the invention, amethod is provided for detecting and measuring antiphospholipidantibodies in a fluid such as human serum or plasma. Serum or plasma isincubated with the phospholipid coated microspheres of the invention atan effective temperature to allow binding of the antiphospholipidantibodies. The microspheres are then contacted with a secondaryantibody directed against human immunoglobulins, this secondary antibodybearing a suitable detectable label, whereby detection and quantitationof the label permits detection and quantitation of the boundantiphospholipid antibodies.

Incubation of sera with phospholipid coated microspheres of theinvention at various temperatures showed that as the temperature ofincubation is increased, non-specific binding of the secondary antibodyalso is increased, as seen in FIG. 1, panel B. In accordance with apreferred embodiment, the incubation is carried out at a temperature inthe range of about 0° C. to about 22° C. An especially preferred rangeis about 0° C. to about 4° C., in which range the non-specific bindingis at a minimum.

At 0° C., the binding reaction approaches saturation at about 30minutes, as seen in FIG. 1, panel A. Saturation is reached more quicklyat higher temperatures.

The preferred pH range for binding of serum antiphospholipid antibodiesto the phospholipid coated beads of the invention is about pH 6.8 toabout pH 7.8. A pH range of about 7.2 to about 7.4 is especiallypreferred.

Icteric and haemolysed serum samples may be assayed by the method of theinvention without problems.

As will be understood by those skilled in the art, a variety oftechniques may be used in the determination of the secondary labelledantibody, including flow cytometry, latex bead agglutination,fluorescence microscopy, latex bead enhanced laser nephelometry andimmuno-dot blotting.

In accordance with a preferred embodiment of the invention, a rapidscreening method is provided for detection of antiphospholipidantibodies in human serum.

Separate portions of microspheres are each coated with one phospholipidto which antibodies are to be detected. The microspheres coated withdifferent phospholipids are mixed and incubated with serum as describedin Example 3.

A polyvalent secondary antibody reacting with IgG, IgM and IgA andbearing a suitable label, e.g., FITC, is added in excess, withoutseparation of the microspheres from the serum. The mixture is incubatedfor 30 minutes at room temperature in the dark then diluted 30-fold withsaline and analysed directly in a flow cytometer, without separation ofthe microspheres from the incubation mixture. Immune complexes betweenthe immunoglobulins of the test serum and the secondary antibody arepresent but are much smaller than the microspheres and can be excludedby electronic gating during the flow cytometer analysis, as will beunderstood by those skilled in the art.

The fluorescence emitted by the microspheres having bound secondaryantibody is detected and indicates the presence of antiphospholipidantibodies in the test serum to any or all of the phospholipids whichwere used to coat the beads. If desired, the amount of fluorescence willprovide a semi-quantitative assessment of the antiphospholipidantibodies present, if appropriate standard curves are prepared.

In accordance with a further preferred embodiment of the invention, ascreening method is provided for detection and identification ofspecific antiphospholipid antibodies in human serum.

Phospholipid coated microspheres of three sizes, coated respectivelywith phosphoinositol, (PI) phosphatidylserine (PS) and cardiolipin (CL),as described in Example 1, are incubated with serum as described inExample 2. The microspheres are separated from the serum and incubatedwith a cocktail of secondary antibodies containing anti-human IgGF(ab')₂ linked to FITC, anti-human IgM linked to phycoerythrin (PE) andanti-human IgA linked to biotin. Secondary antibodies are titrated to besaturating and incubation is carried out at room temperature in the darkfor 15 to 60 minutes depending on the degree of saturation. Unboundsecondary antibody is removed by centrifugation and the microspheres areresuspended in saturating avidin linked to PE/Texas Red and incubated inthe dark at room temperature for 15 minutes, to allow binding to theIgA-linked biotin.

The microspheres are then separated by centrifugation and resuspended inan iso-osmolar salt solution such as ISOTON II™ suitable for flowcytometry.

Flow cytometric analysis is conducted as in Example 2. The detectedantiphospholipid antibodies can be identified as anti-PI, anti-PS andanti-CL and classified as IgA, IgG or IgM. If desired, the amount offluorescence will provide a semi-quantitative assessment of theantiphospholipid antibodies present, if appropriate standard curves areprepared.

As will be appreciated by those skilled in the art, this screeningprocedure may be carried out using microspheres coated with otherphospholipids, as desired. Furthermore, the procedure is not limited tothree sizes of microspheres, thereby providing a screen for three typesof phospholipid. Additional sizes of microspheres with additionalphospholipids may be employed, as permitted by the discriminatorycapabilities of the flow cytometer used.

It has, however, been found convenient to use three sizes ofmicrospheres of diameters as described in Example 1, as these sizes maybe examined simultaneously within the detection limits of the flowcytometer.

The different size classes of coated beads are mixed in proportions suchthat the surface area presented by each population to the test serum wasthe same.

It has been found that when whole molecule IgG is used as secondaryantibody, either alone or in a cocktail with other antibodies,non-specific binding tends to be high and interferes with the flowcytometric analysis. For determination of IgG antibodies, it ispreferred to use the F(ab')₂ fragment of human IgG as secondaryantibody.

Human serum may be screened first by the rapid method to eliminatenegatives before screening to identify particular antiphospholipidantibodies or may be screened directly with the more discriminatingscreening procedure.

Other suitable detectable labels for the second antibodies will be knownto those skilled in the art and include fluorescent compounds such asfluoresceins, rhodamines, erythrosin, phycoerythrin, phycoerythrin/Texasred conjugate, Texas red and propidium iodide. For assays such asimmunodot blot, lables such as peroxidase, alkaline phosphatase andferritin may be used.

In accordance with a further embodiment of the invention, a method isprovided for quantitation of antiphospholipid antibodies in human serum.

For optimal quantitation of antiphospholipid antibodies, it is preferredto use phospholipid coated microspheres of one size and phospholipidtype and to use secondary antibody of one immunoglobulin class labelledwith a convenient fluorophore such as FITC or PE in the method of theinvention.

Standard curves are prepared using commercially availableantiphospholipid antibody standards incubated with microspheres coatedwith the appropriate phospholipid and then reacted with known amounts ofthe appropriate immunoglobin class as secondary antibody, as describedin Example 4.

Sera from patients presenting with thrombotic tendencies have beenanalysed by the method of the invention and antiphospholipid antibodieshave been detected in over half of them.

The serum levels of anti-cardiolipin antibodies have been determined ina series of thrombotic patients both by an ELISA technique and by themethod of the invention. As seen in FIG. 7, the method of the inventionprovides comparable sensitivity to presently available ELISA techniques.

The assay methods have been described with reference to serum but itwill be understood by those skilled in the art that plasma may besimilarly assayed.

In accordance with a further embodiment of the invention, a convenientand rapid method is provided for isolating antiphospholipid antibodies.

A solution containing antiphospholipid antibodies to be isolated ismixed with microspheres coated with the appropriate phospholipid orphospholipids and blocked as in Example 1, and the mixture is stirred atabout 0° C. for one hour. The beads are pelleted by centrifugation andwashed twice with 0.01M Tris, 0.14M NaCl (pH 7.35) containing 10% v/vfetal calf serum. The beads are washed once with 0.01M Tris, 0.14M NaCl(pH 7.35) and centrifuged to a pellet. The bound antibodies are elutedwith a chaotropic salt such as KI, KSON or NaI (1M) with incubation atroom temperature for 1 hour. The phospholipid beads are then removed bycentrifugation and the eluted antibodies subjected to furtherpurification on a Protein A column or hydrazide column composed ofantibodies to the desired immunoglobulin subclass.

In accordance with a further embodiment of the invention, a method isprovided for raising antibodies to a phospholipid.

Various immunisation schemes have been employed to raise antibodies tophospholipids but these have generally yielded non-specific antibodies.

The phospholipid coated microspheres of the invention presentphospholipid antigens in such a way that they can bind to their specificantibodies. Administration of these phospholipid microspheres to ananimal, for example by subcutaneous injection, conveniently presents theanimal's immune system with a phospholipid antigen against whichspecific antibodies are elicited. The coated microspheres present alarge surface area displaying antigen and adapted to elicit a goodimmunological response. The microspheres are coated with phospholipid asin Example 1 but are not treated with blocking agent.

In accordance with a further embodiment, kits for determiningantiphospholipid antibodies are provided comprising phospholipid coatedparticles and one or more labelled reagents capable of binding to theantiphospholipid antibodies.

The following examples are given for the purpose of illustrating theinvention and the present invention is not limited thereto.

Materials

The following materials were purchased from Sigma Chemicals (St. Louis,Mo.): phosphatidylinositol, phosphatidylserine, cardiolipin, goatanti-human IgG F(ab')₂ linked to fluoresceinisothyocyanate (FITC), tris(hydroxymethyl) methylamine, sodium chloride. Heat inactivated fetalcalf serum was purchased from GIBCO (Burlington, Ont.). Avidin linked toPE/Texas Red was purchased from Southern Biotechnology (Birmingham,Ala.). Polystyrene microspheres (beads) were purchased from PolySciences(Warrington, Pa.). Absolute ethanol was produced by triple distillationof stock ethanol purchased from BDH (Edmonton, Alta). Chloroform andmethanol were purchased from BDH (Edmonton, Alta). Isoton II waspurchased from Coulter Electronics (Hialeah, Fla.). Flow cytometricanalysis was performed on a FACScan from Becton Dickinson (Mountainview,Calif.). Anti-phospholipid antibody standards were purchased fromAntiphospholipid Antibody Associates (Louisville, Ky.).

EXAMPLE 1 Phospholipid Preparation

Cardiolipin (CL) was supplied as a 5 mg/mL solution in ethanol and usedwithout further modification. Phosphatidylinositol (PI) was initiallydissolved in chloroform:methanol (99:1) then further diluted in absoluteethanol to produce a stock solution of 10 mg/mL. Phosphatidylserine (PS)was initially dissolved in chloroform:methanol (95:1) then furtherdiluted in absolute ethanol to produce a stock solution of 25 mg/mL. Allsolutions were stored in the dark at 4° C.

Pre-treatment of Microspheres

Polystyrene microspheres (beads) of defined size were supplied by themanufacturer in sterile distilled water. Prior to coating withphospholipid, the beads were washed several times with distilled waterfollowed by several washes in absolute ethanol. Washing was accomplishedby centrifugation. All ethanol washes and subsequent manipulation ofbeads should be carried out in glass vessels using glass transferimplements.

Coating Procedure

After washing, beads were resuspended to a final volume of 1 mL inabsolute ethanol.

Beads of three different sizes were coated with three differentphospholipids; beads of average diameter 5.8 μm (±0.1 μm) were coatedwith CL, beads of 2.9 μm (±0.1 μm) with PS and beads of 1.6 μm (±0.1 μm)with PI.

The polystyrene bead number was adjusted so that the surface areapresented by each size of bead to be coated was the same. In a likemanner, the phospholipid concentrations were adjusted to provideapproximately similar amounts by weight per unit area of bead. Table 1shows an example of suitable coating parameters.

                  TABLE 1                                                         ______________________________________                                        PHOSPHOLIPIDS                                                                 (BEAD DIAMETER)                                                                            Pl (1.6 μm)                                                                          PS (2.9 μm)                                                                           CL (5.8 μm)                              ______________________________________                                        Particles per mL                                                                           1.11 × 10.sup.10                                                                  0.18 × 10.sup.10                                                                   0.02 × 10.sup.10                      Particles coated (100 μm)                                                               11.1 × 10.sup.8                                                                   1.80 × 10.sup.8                                                                    2.00 × 10.sup.8                       Surface Area (×10.sup.10 μm.sup.2)                                                0.89      0.48       0.22                                        Coating Volume                                                                             40 μL  9 μL    20 μL                                    Phospholipid Conc.                                                                         10 mg/mL  25 mg/mL   5 mg/mL                                     (Stock solution)                                                              Coating/Unit Area                                                                          0.45      0.47       0.45                                        (mg/10.sup.10 μm.sup.2)                                                    ______________________________________                                    

The volumes of phospholipid indicated in Table 1 (relative phospholipidconcentrations also given in Table 1) were added to the bead suspensionsand mixed by aspiration. The bead suspensions were sealed and incubatedovernight at 4° C. in the dark.

The phospholipid coated beads were then treated with a blocking agent toreduce non-specific binding of immunoglobulins and other potentiallyinterfering proteins. Phospholipid coated beads in ethanol were allowedto come to room temperature and were washed by mixing gently byaspiration with an equal volume of Tris/NaCl buffer (0.01M Tris, 0.14MNaCl, pH 7.2), followed by centrifugation. After a further two washeswith buffer, the beads were resuspended in "blocking solution" (500 μL),i.e. 10% v/v fetal calf serum in 0.01M Tris, 0.14 NaCl, pH 7.2, andincubated at 37° C. for 30 minutes followed by rapid cooling at 0° C.

Once the phospholipid-coated beads have been blocked, it is no longernecessary to use glass transfer implements and glass vessels. Reactionsare conveniently carried out in 1.5 mL polypropylene microfuge tubes.

The un-blocked phospholipid coated beads may be stored for later usebefore treatment with the blocking agent. Various storage conditionswere examined, using cardiolipin coated beads, and some examples ofsuitable storage conditions are shown in Table 2.

                  TABLE 2                                                         ______________________________________                                        STORAGE CONDITIONS                                                                          STORAGE TIME                                                                              BETWEEN RUN CV                                      ______________________________________                                        4° C., in                                                                            4 months     7% (N = 10)                                        Phospholipid/                                                                 EtOH Solution, in                                                             Dark                                                                          -20° C., freeze                                                                      4 months    10% (N = 10)                                        dried, in dark                                                                -20° C., in glycerol,                                                                2 months    15% (N = 7)                                         in dark                                                                       ______________________________________                                    

EXAMPLE 2

Beads of three sizes were coated with CL, PS and PI as described inExample 1 and mixed in the proportions 1 part 1.6 μm beads: 2 parts 2.9μm beads:4 parts 5.8 μm beads, based on the number of particles coatedfor each bead size as in Table 1. The bead mixture was suspended inblocking solution.

For assay, 5 μL of serum to be analysed was mixed with 495 μL beadmixture suspension and incubation was carried out at 0° for 60 minutes.Unbound immunoglobulins and other contaminating proteins were removedfrom the beads by centrifugation and the beads were resuspended in asecondary antibody mixture containing goat anti-human IgG-FITC F(ab')₂,goat anti-human IgM-PE and goat anti-human IgA-biotin. Saturatingconcentrations of antibodies were used, as determined by titration.After a 30 minute incubation at room temperature, the beads wereseparated from unbound antibodies by centrifugation and resuspended insaturating concentrations of avidin linked to PE/Texas red, withincubation at room temperature in the dark for 15 minutes. Beads wereagain separated by centrifugation and resuspended in balanced saltsolution, ISOTON II™ (Coulter Electronics).

Resuspension volumes of 0.5 to 1.0 mL were employed for use with aFACScan flow cytometer for analysis. The resuspended beads weretransferred to 12×75 mm polystyrene tubes and analyzed by standard flowcytometric methods. The flow cytometer was capable of measuring five (5)separate parameters on each particle examined. Forward scatter 180°relative to the incident laser light (488 nm), indicated the size of theparticle. Side scatter, 90° relative to the incident laser, indicatedinternal complexity of the particle. In addition three differentfluorescence parameters, based on emission spectra (post excitation with488 nm laser), were analyzed. Spectral overlap by the three availablefluorescent tags (FITC, PE, PE/Texas Red) was compensated forelectronically as outlined by the manufacturer. All data was stored todisk in list-mode. Subsequently, the data were analyzed for each beadsize based on electronically selecting (gating) each bead population andanalyzing the fluorescence exhibited.

The machine was calibrated daily using fluorescent microparticles(Calibrite Beads, Becton Dickinson) and compensation for interchannelbleeding was set for three colour analysis where more than one labelledantibody was measured.

The excellent discrimination between the three bead sizes coated withPI, PS and CL respectively is shown in FIG. 1, a forward scatterhistogram.

EXAMPLE 3

Sera to be screened were diluted with bead mixture suspension as inExample 2 and incubated at 0° C. for 60 minutes.

A saturating concentration of goat anti-human polyvalent (IgA, IgM, IgG)antiserum labelled with FITC was added directly to the serum/beadmixture, without separation of beads from test serum and incubation wascarried out at room temperature in the dark for 30 minutes.

A negative control and a positive control were included in each analysisas a check on run to run reproducibility.

After incubation, the incubation mixture was diluted about 30-fold withsaline and was directly analysed by flow cytometry as in Example 2.

EXAMPLE 4

For quantitation of serum antibodies of a particular immunoglobulinclass directed against a specific phospholipid, test sera were analysedby the procedure described in Example 2 employing beads of one sizecoated with the appropriate phospholipid and using as secondary antibodyFITC or PE labelled goat anti-human antibodies against IgM or IgA, orgoat anti-human IgG-FITC F(ab')₂.

Standard curves were prepared using antiphospholipid antibody standardsobtained from Antiphospholipid Antibody Associates. The peak meanchannel fluorescence was plotted logarithmically against the log of theantiphospholipid antibody concentration (in GPL, MPL or APL units).

Concentration of antiphospholipid antibody was expressed asimmunoglobulin binding units to phospholipid, abbreviated as GPL, MPLand APL for IgG, IgM and IgA bound to phospholipid respectively.

The immunoglobulin binding units were defined by Harris et al (Clin.Exp. Immunol., 69: 215, 1987) to be the phospholipid binding activity of1 μg/mL of an affinity purified IgG (GPL), IgM (MPL) or IgA (APL)preparation from a standard serum.

Examples of standard curves generated are shown in FIGS. 3 and 4.

EXAMPLE 5

Serum samples were obtained from 26 healthy volunteers and levels ofantiphospholipid antibodies to PI, PS and CL were measured as in Example4. Polyvalent secondary antibodies were employed, giving a measure oftotal IgG, IgM and IgA against each phospholipid. The results obtainedare shown in FIG. 5.

EXAMPLE 6

Serum samples were obtained from eight patients with a history ofthrombotic episodes. An initial screen was performed by the procedure ofExample 2 to determine the immunoglobulin class and phospholipidspecificity of any antiphospholipid antibodies present. Each type ofantiphospholipid antibody detected by the screen was then quantitated asdescribed in Example 4. Antibodies to one or more phospholipids werefound in seven of the eight patients, as seen in FIG. 6. The results arepresented as negative, low positive, medium positive and high positiveas recommended by Harris et al (Clin, Exp. Immunol., 68: 215, 1987). Thedegree of positivity was based on the number of standard deviationsabove the normal ranges of 26 normal controls as follows: Negative≦3 sd,Low Positive=between 3 and 5 sd, Medium Positive=between 5 and 7 sd andHigh Positive>7 sd; as regards mean channel fluorescence.

EXAMPLE 7

A comparison was made of serum anti-cardiolipin antibody levelsdetermined by the method of the invention and those obtained by theELISA method of Loizou et al (Clin. Exp. Immunol., (1985) vol. 62, pp738-745). Sera examined were obtained from 30 patients with thrombotictendencies.

After screening the sera as in Example 2 for the presence ofanti-cardiolipin antibodies, levels of GPL were determined in positivesera by the procedure of Example 4. Binding of GPL was assessed and GPLvalues were determined based on the cut-off values recommended byAntiphospholipid Antibody Associates. For the ELISA method, thesecondary antibody used was goat anti-human IgG linked to peroxidase.The results are shown in FIG. 7.

Although only certain embodiments of the present invention have beendescribed and illustrated, the present invention is not limited to thefeatures of these embodiments, but includes all variations andmodifications within the scope of the claims.

We claim:
 1. A method for determining the presence, in a fluid, ofanti-target-phospholipid antibodies, comprising:contacting microsphereswith a solution of a target phospholipid, which is selectively bound byantibodies to be determined, under conditions effective to allow coatingof the phospholipid on the microspheres thereby forming asolution/phospholipid-coated microsphere mixture; washing thephospholipid-coated microspheres by mixing thesolution/phospholipid-coated microsphere mixture with an equal volume ofaqueous buffer and collecting the washed phospholipid-coatedmicrospheres; contacting a fluid suspected of containinganti-target-phospholipid antibodies with the phospholipid-coatedmicrospheres to permit binding of any anti-target-phospholipidantibodies present in the fluid to the phospholipid-coated microspheres;and determining the presence of any anti-target phospholipid antibodiesbound to the phospholipid-coated microspheres.
 2. The method of claim 1,wherein the microspheres comprise beads.
 3. The method of claim 1,wherein the microspheres are selected from the group consisting ofpolypropylene, polyethylene, acrylonitrile, polystyrene, polycarbonate,and nitrocellulose.
 4. The method of claim 1, wherein the microspherescomprise latex microspheres.
 5. The method of claim 1, wherein themicrospheres comprise magnetic microspheres.
 6. The method of claim 1,wherein the phospholipid is selected from the group consisting ofcardiolipin, phosphatidyl inositol, phosphatidyl serine, phosphatidicacid, and phosphatidyl ethanolamine, and mixtures of the phospholipids.7. The method of claim 1, wherein the fluid is a biological fluid. 8.The method of claim 7, wherein the biological fluid comprises serum orplasma.
 9. The method of claim 8, wherein the serum or plasma compriseshuman serum or plasma.
 10. The method of claim 1, wherein anyanti-target-phospholipid antibodies bound to the phospholipid-coatedmicrospheres are determined with a detectably-labeled reagent whichbinds to the anti-target-phospholipid antibodies.
 11. The method ofclaim 10, wherein the detectably-labeled reagent comprisesdetectably-labeled polyvalent anti-human immunoglobulin antiserum. 12.The method of claim 10, wherein the detectably-labeled reagent comprisesat least one anti-human monovalent antibody or binding fragment thereofdirected against at least one immunoglobulin class.
 13. The method ofclaim 10, wherein the detectably-labeled reagent comprises a detectablelabel attached to anti-immunoglobulin antibodies or binding fragmentsthereof selected from the group consisting of anti-human IgG, anti-humanIgM and anti-human IgA antibodies and the presence of anyanti-target-phospholipid antibodies is determined as anti-phospholipidIgG, IgM or IgA antibodies.
 14. The method of claim 10, wherein thedetectably-labeled reagent comprises a detectable label attached toanti-immunoglobulin antibodies or binding fragments thereof selectedfrom the group consisting of anti-human IgG F(ab')₂, anti-human IgA andanti-human IgM and the presence of any anti-target-phospholipidantibodies is determined as anti-phospholipid IgG, IgM or IgAantibodies.
 15. The method of claim 14, wherein the detectably-labeledreagent comprises anti-human IgG F(ab')₂ -FITC, anti-humanIgM-phycoerythrin and anti-human IgA-biotin, and the method furthercomprises contacting the microspheres with avidin-Texas red to permitbinding to the biotin.
 16. The method of claim 1, wherein theanti-target-phospholipid antibodies comprise anti-target-phospholipidantibodies associated with arterial and venous thrombosis, recurrentfetal loss, thrombocytopenia, pulmonary embolism, coronary thrombosis,cerebral thrombosis, livedo reticularis or HIV infection.
 17. A methodfor separately determining the presence, in a fluid, of at least twodifferent anti-target-phospholipid antibodies which selectively binddifferent target phospholipids, comprising:obtaining microspheres of atleast two size classes; separately contacting each microsphere classwith a solution of a different target phospholipid that is selectivelybound by antibodies to be determined, under conditions effective toallow coating of each microsphere class with a different targetphospholipid, thereby forming at least two differentsolution/phospholipid-coated microsphere mixtures; washing eachmicrosphere class by mixing each solution/phospholipid-coatedmicrosphere mixture with an equal volume of aqueous buffer andcollecting each of the washed phospholipid-coated microsphere classes;contacting a fluid suspected of containing anti-target-phospholipidantibodies with a mixture comprising phospholipid-coated microspheresfrom each of the at least two classes, and allowing anyanti-target-phospholipid antibodies present in the fluid to bind to thephospholipid-coated microspheres; and determining the presence of anyanti-target-phospholipid antibodies bound to each phospholipid-coatedmicrosphere class.
 18. The method of claim 17, wherein the fluidcomprises a biological fluid.
 19. The method of claim 18, wherein thebiological fluid comprises serum or plasma.
 20. The method of claim 19,wherein the serum or plasma comprises human serum or plasma.
 21. Themethod of claim 17, wherein any anti-target-phospholipid antibodiesbound to each phospholipid-coated microsphere class are separatelydetermined by flow cytometry with a detectably-labeled reagent, whichbinds to the at least two different anti-target-phospholipid antibodies.22. The method of claim 21, whereinthe microsphere classes comprisefirst, second and third microsphere size classes; the first classcomprises microspheres of about 1.6 μm average diameter; the secondclass comprises microspheres of about 2.9 μm average diameter; the thirdclass comprises microspheres of about 5.8 μm average diameter; and eachmicrosphere class is coated with a different target phospholipidselected from the group consisting of cardiolipin, phosphatidylinositol, phosphatidyl serine, phosphatidic acid andphosphatidylethanolamine.
 23. The method of claim 22, wherein thedetectably-labeled reagent comprises a separately-detectable labelattached to anti-immunoglobulin antibodies selected from the groupconsisting of anti-human IgG F(ab')₂, anti-human IgM and anti-human IgAand the presence of any anti-target-phospholipid antibodies isdetermined as anti-phospholipid IgG, IgM or IgA antibodies.
 24. Themethod of claim 17, wherein the anti-target-phospholipid antibodiesdetermined comprise anti-target-phospholipid antibodies associated witharterial and venous thrombosis, recurrent fetal loss, thrombocytopenia,pulmonary embolism, coronary thrombosis, cerebral thrombosis, livedoreticularis or HIV infection.
 25. A method for determining the amount,in a fluid, of anti-target-phospholipid antibodies, comprising:(a)contacting microspheres with a solution of a target phospholipid, whichis selectively bound by antibodies to be determined, under conditionseffective to allow coating of the phospholipid on the microspheresthereby forming a solution/phospholipid-coated microsphere mixture; (b)washing the phospholipid-coated microspheres by mixing thesolution/phospholipid-coated microsphere mixture with an equal volume ofaqueous buffer, and collecting the washed phospholipid-coatedmicrospheres; (c) separately contacting at least two portions of thephospholipid-coated microspheres with at least two known amounts ofstandard anti-target-phospholipid antibodies to permit binding of thestandard anti-target-phospholipid antibodies to the phospholipid-coatedmicrospheres, and contacting the standard anti-target-phospholipidantibodies bound to the phospholipid-coated microspheres with adetectably-labeled reagent which binds to anti-target-phospholipidantibodies to provide a standard curve; (d) contacting a fluid suspectedof containing anti-target-phospholipid antibodies with a further portionof phospholipid-coated microspheres to permit binding of anyanti-target-phospholipid antibodies present in the fluid to thephospholipid-coated microspheres and contacting anyanti-target-phospholipid antibodies bound to the phospholipid-coatedmicrospheres with the detectably-labeled reagent which binds toanti-target-phospholipid antibodies; and (e) determining the amount ofanti-target-phospholipid antibodies present in the fluid by comparingthe amount of detectably-labeled reagent bound to thephospholipid-coated microspheres of step (d) with the standard curve ofstep (c).